The present invention relates to a novel method of recombinantly producing, recovering and purifying angiostatin protein (EntreMed Inc., Rockville, Md.).
Angiostatin is a protein which is a potent inhibitor of developing blood vessels and tumor growth. Angiostatin is believed to play an important role in the inhibition of the development of blood vessels to new tumor metastases.
Isolation and purification of proteins, such as angiostatin protein, in high yield from biological material, such as tissue extracts, cell extracts, broth from incubation systems, and culture medium is often frought with problems in view of the numerous proteins and other undesirable molecules present in an homogenate or extract. What is needed are recombinant methods of producing angiostatin protein that will provide the large amounts of angiostatin protein required for clinical use, including, but not limited to, cancer therapy. Such methods should produce angiostatin protein in an efficient and convenient manner in a culture broth which is amenable to procedures designed to recover and purify angiostatin protein in high yields. Separating a, specific protein of interest from potential contaminants presents a challenge in view of numerous factors, such as contamination of cellular homogenates with proteolytic enzymes that may digest the protein. Other undesirable cellular constituents that may be present in homogenates, include but not limited to, pigments, cytochromes, lipids, free radicals, oxidases and other lysosomal enzymes, and oxides. Some of these substances may affect the protein of interest by stripping electrons, affecting disulfide bonds and changing the conformation of the protein.
Centrifugation of cells, including yeast, bacteria, insect and other cells used for recombinant production of proteins, such as angiostatin protein, could possibly result in damage to the cells with concomitant release of undesirable biological material. What is needed is a method for recovery and purification of protein, such as angiostatin protein, which does not employ centrifugation.
Methods for recombinant production, recovery and purification of angiostatin protein on a large scale are required to produce and isolate the amounts of purified angiostatin protein needed for administration to patients and also for research purposes.
Also needed is a method for purifying recombinantly-produced angiostatin protein which avoids the need for centrifugation of the culture broth, thereby avoiding problems associated with cell lysis. This method should be capable of use on a large scale to recover and purify angiostatin protein in quantities needed for clinical administration and research.
What is also needed is a method for purifying recombinantly produced angiostatin protein which minimizes contamination with cytochromes, pigments, enzymes, and other undesirable cellular constituents.
Also needed are solutions for storage of angiostatin protein following the recovery and purification process which optimizes solubility properties of angiostatin protein.
The present invention solves these problems inherent in the recovery and purification of proteins, particularly angiostatin protein, by providing new and useful methods for recombinant production, recovery and purification of proteins, especially angiostatin protein. The present invention provides new and useful methods for recombinantly producing angiostatin protein in large amounts. The present invention provides a method for recovery and purification of angiostatin protein. The present invention also provides new and useful solutions for storage of angiostatin protein. These methods provide the benefit of preserving the biological activity of angiostatin protein. Preservation of the biological activity of angiostatin protein is crucial for administration of angiostatin protein to humans and animals for the purpose of inhibition of undesirable angiogenesis, for other biological activities, and for research investigations or other types of biological testing.
Angiostatin protein is effective in treating diseases or processes that are mediated by, or involve, angiogenesis. The angiogenesis mediated diseases include, but are not limited to, solid tumors; blood born tumors such as solid tumors, blood borne tumors, leukemias; tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, colon cancer, retrolental fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation.
In one embodiment, the present invention provides new and improved methods for recombinant production of biologically active angiostatin protein in high yield.
In another embodiment, the method of the present invention is useful for recovery and purification of recombinantly-produced angiostatin protein.
In another embodiment, the method of the present invention is useful for recovery and purification of angiostatin protein from extracts of biological fluids, cells and tissues.
An advantage of the present invention is that higher amounts of biologically active angiostatin protein are recombinantly produced. Another advantage of the present invention is that greater amounts of angiostatin protein are recovered than obtained with prior art methods. Yet another advantage of the present invention is that higher yields of more purified, and biologically active angiostatin protein are obtained. Still another advantage of the present invention is that angiostatin protein may be stored in buffers for extended periods of time, and also subjected to lyophilization, while preserving biological activity. An advantage of the present invention is that it permits angiostatin protein to be stored in vials or other containers, either in a solution which may be liquid or frozen, or lyophilized, and optionally shipped to a recipient.
Accordingly, an object of the present invention is to provide an improved method for recombinant production of large amounts of biologically active angiostatin protein.
Another object of the present invention is to provide a method for recovery and purification of recombinantly produced proteins.
Yet another object of the present invention is to provide a method for recovery and purification of angiostatin protein.
Another object of the present invention is to provide a method for recovery and purification of angiostatin protein, particularly recombinantly produced angiostatin protein.
An advantage of the purification methods of the present invention is that undesirable proteins, lipids and pigments are efficiently separated from the desired protein, especially angiostatin protein.
It is another object of the present invention to provide solutions which provide favorable solubility conditions for angiostatin protein, particularly recombinantly-produced angiostatin protein while retaining biological activity of angiostatin protein.
Another advantage of the methods of the present invention is that centrifugation of the broth from fermentation steps in recombinant production of angiostatin protein is avoided, thereby preventing unwanted cellular lysis and potential contamination of angiostatin protein with additional proteins, pigments, enzymes and other cellular chemicals and debris.
Another object of the present invention is to provide methods amenable to large scale production, recovery and purification of recombinantly-produced angiostatin protein.
Another advantage of the present invention is that the recovered and purified angiostatin protein is provided in a solution which optimizes solubility of angiostatin protein, while preserving the bioactivity of angiostatin protein.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description.